Journal: Veterinary Sciences

Article Title: Roles of WNT6 in Sheep Endometrial Epithelial Cell Cycle Progression and Uterine Glands Organogenesis

doi: 10.3390/vetsci8120316

Figure Lengend Snippet: Details of antibodies.

Article Snippet: β-catenin , 51067-2-AP , Proteintech, Wuhan, China , _ , 1:5000 , β-Catenin, also known as CTNNB1, is a key downstream component of the canonical Wnt pathway that plays diverse and critical roles in embryonic development and adult tissue homeostasis..

Techniques: Quantitative Proteomics, Transformation Assay, Gene Expression

Journal: Veterinary Sciences

Article Title: Roles of WNT6 in Sheep Endometrial Epithelial Cell Cycle Progression and Uterine Glands Organogenesis

doi: 10.3390/vetsci8120316

Figure Lengend Snippet: The effects of WNT6 transfection on β-catenin expression in EECs. ( A , B ) Relative expression of WNT6 and β-catenin protein to β-actin by WNT6 knockdown. ( C , D ) Relative expression of WNT6 and β-catenin protein to β-actin by WNT6 overexpression. si-WNT6, the small interfering RNA for WNT6 . pEX-4-WNT6, pEX-4 vector with WNT6 -CDs inserted. siRNA with scrambled sequence absent in the sheep genome and pEX-4 was severed as negative control for knockdown and overexpression, respectively. The values are presented as means ± SDs. Means with different letters indicate significant differences ( p < 0.05).

Article Snippet: β-catenin , 51067-2-AP , Proteintech, Wuhan, China , _ , 1:5000 , β-Catenin, also known as CTNNB1, is a key downstream component of the canonical Wnt pathway that plays diverse and critical roles in embryonic development and adult tissue homeostasis..

Techniques: Transfection, Expressing, Knockdown, Over Expression, Small Interfering RNA, Plasmid Preparation, Sequencing, Negative Control

Journal: Royal Society Open Science

Article Title: Dendropanax morbiferus leaf extract facilitates oligodendrocyte development

doi: 10.1098/rsos.190266

Figure Lengend Snippet: Dendropanax morbiferus leaf extracts downregulated inhibitory factors and upregulated promoting factors for OL differentiation in pure OPC cultures. ( a – d ) OPCs were treated with EtOH- or DM leaf extract-containing medium during DIV 1–3 and were used for RNA extraction and cDNA synthesis, followed by real-time PCR to ascertain the relative expression pattern of the following genes: ( a ) Olig1 , ( b ) ID2 , ( c ) Ascl1 and ( d ) MBP . The expression of Olig1 and ID2 but not Ascl1 was significantly downregulated, while the expression of MBP was in a tendency to increase by DM leaf extract treatment. Bars represent mean ± s.e.m. * p < 0.05; ** p < 0.01; n = 3 experiments, Student's t -test. ( e ) Three different cultures were pooled and used for western blot analysis to detect MBP and phospho-p42/44. β-actin was used for normalization. Compared with EtOH-treated cultures, upregulations in MBP ( f ) and in phosphorylation of p42/44 ( g ) were observed in the DM leaf extract-treated cultures.

Article Snippet: Rat antibody against MBP (1 : 500, MAB386, Chemicon), rabbit antibodies against phospho-p42/44 (1 : 1000, 4370P, Cell signaling), and β-actin (1 : 5000, Abcam) were used for immunoblotting.

Techniques: RNA Extraction, cDNA Synthesis, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Phospho-proteomics

Journal: iScience

Article Title: Bombinin-BO1 induces hepatocellular carcinoma cell-cycle arrest and apoptosis via the HSP90A-Cdc37-CDK1 axis

doi: 10.1016/j.isci.2024.110382

Figure Lengend Snippet:

Article Snippet: Materials purchased from Proteintech Group (Wuhan, China) included antibodies against GAPDH (proteintech, 10494-1-AP, 1:5000), Beta Actin (proteintech, 66009-1-Ig, 1:1000), goat anti-rabbit IgG (proteintech, 66912-1-lg, 1:10000), Pro Caspase-8 (Abcam, ab108333, 1:10000), Caspase-9 (proteintech, 10380-1-AP, 1:1000), Cleaved Caspase-9 (Abcam, ab2324, 1 ug/mL), caspase-3 (proteintech, 19677-1-AP, 1:2000), cleaved caspase-3 (Abcam, ab2302, 1:100), Bcl-2 (proteintech, 12789-1-AP, 1:1000), Bax (proteintech, 50599-2-lg,1:1000), HSP90A (proteintech, 60318-1-Ig, 1:5000), Cdc37 (proteintech, 10218-1-AP, 1:200), CDK1 (proteintech, 19532-1-Ig, 1:200), Cyclin A2 (proteintech, 18202-1-AP, 1:5000).

Techniques: Recombinant, Magnetic Beads, RNA Extraction, Quantitation Assay, Protein Concentration, Software, Lysis

Journal: eLife

Article Title: The nanoscale organization of the Nipah virus fusion protein informs new membrane fusion mechanisms

doi: 10.7554/elife.97017

Figure Lengend Snippet: Figure 4. The distribution and organization of NiV-F constructs in VLPs. (A) The incorporation of F-WT and mutants in VLPs. NiV-M-GFP, G-HA, and FLAG-tagged F-WT or mutants were transfected to 293T cells. The supernatants were collected at 48 hr post-transfection and analyzed on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) followed by western blotting. NiV-M was probed by polyclonal goat anti-GFP, NiV-G polyclonal rabbit anti-HA, F0 and F2 M2 monoclonal mouse anti-FLAG antibody. (B) Cross-section (Δz = 100 nm) of single-molecule localization microscopy (SMLM) images of the FLAG-tagged NiV-F-WT (WT), L53D, V108D, and Q393L on individual VLPs. Scale bar: 0.2 μm. (C) The classification of the ordered sequence of reachability distances of the NiV-F constructs localizations. Orange: F-WT (n = 306) and Q393L (n = 323); blue: L53D (n = 310) and V108D (n = 329). n is the number of VLPs used for classification analysis.

Article Snippet: DOI: https://doi.org/10.7554/eLife.97017 16 of 23 Reagent type (species) or resource Designation Source or reference Identifiers Additional information Chemical compound, drug HEPES Fisher Scientific 15- 630- 080 25 mM for fusion buffer Chemical compound, drug Glutamine Fisher Scientific 25- 030- 081 2 mM for fusion buffer Chemical compound, drug CaCl2 Sigma- Aldrich C7902 1 mM for fusion buffer Chemical compound, drug Probenecid Sigma- Aldrich P8761- 25G 2.5 mM for fusion buffer Chemical compound, drug Solution D Fisher Scientific K1156 1:20 for Entry kinetics loading solution Antibody Anti- FLAG mouse monoclonal Sigma- Aldrich F1804 IF, SMLM: 1:100, Flow: 1:200; WB: 1:500–1:5000 Antibody Anti- HA rabbit polyclonal antibody Biolegend 902301 IF: 1:900 WB: 1:2000 Antibody Anti- GFP goat polyclonal antibody Abcam Ab5450 WB: 1:1000 Antibody Anti- mCherry rabbit polyclonal antibody Abcam Ab167453 WB: 1:2000 Antibody Anti-β-lactamase mouse monoclonal Santa Cruz Biotechnology Sc- 66062 WB: 1:1000 Antibody Anti- mouse donkey polyclonal antibody, Alexa Fluor 647 conjugated Invitrogen A31571 IF and SMLM: 1:400 Antibody Anti- rabbit donkey polyclonal antibody, Alexa Fluor 488 conjugated Invitrogen A21206 IF: 1:400 Antibody Anti- mouse donkey polyclonal antibody, Alexa Fluor 488 conjugated Invitrogen A21202 Flow: 1:400 Antibody Anti- goat donkey polyclonal antibody, HRP conjugated Jackson Immunoresearch 705- 035- 147 WB: 1:5000 Antibody Anti- mouse goat polyclonal antibody, HRP conjugated Bio- Rad 1705047 WB: 1:5000 Antibody Anti- rabbit goat polyclonal antibody, HRP conjugated Bio- Rad 1706515 WB: 1:5000 Commercial assay or kit μMACS anti- DYKDDDDK starting kit Miltenyi Biotec 130- 101- 636 Commercial assay or kit Renilla Luciferase Assay System Promega E2820 Commercial assay or kit LiveBLAzer FRET- B/G Loading Kit with CCF2- AM Invitrogen K1032 Commercial assay or kit QIAamp Viral RNA Kits for RNA Extraction QIAGEN 52904 Commercial assay or kit SuperScript III First- Strand Synthesis System Invitrogen 18080051 Software SMLM image reconstruction Liu et al., 2018 MATLAB Codes are available upon request.

Techniques: Construct, Transfection, Polyacrylamide Gel Electrophoresis, SDS Page, Western Blot, Microscopy, Sequencing

Journal: Science Progress

Article Title: Role of thrombus-derived exosomal lncRNA LOC101928697 in regulating endothelial function via FUS protein interaction in myocardial infarction

doi: 10.1177/00368504251372111

Figure Lengend Snippet: Analysis of the correlation between FUS protein and myocardial infarction. (a) Enrichment Analysis Bar Plot based on differential gene expression profiles in lncRNA microarray analysis.(b) Detection information about lncRNA LOC101928697 binding to FUS proteins in AnnoLnc2 database. (c) Detection information about lncRNA LOC101928697 binding to FUS protein in RBPDP database. (d) Scores in the RPISeq database on the model of lncRNA LOC101928697 binding to FUS protein. (e-g) Prediction information about lncRNA LOC101928697 binding to FUS protein in catRAPID website, (e) Statistical map information about protein and RNA binding sites, (f) Total scoring information, and (g) Interaction map showing the interaction region between protein and RNA. (h-i) Analyses about bioinformatics techniques based on GSE163772 in the GEO database, where (h) is a statistical map of FUS gene expression in endothelial cells of a mouse model of myocardial infarction, and (i) A scatter plot about the correlation between the level of FUS gene expression and the disease state (control vs. myocardial infarction).

Article Snippet: After extensive washing, the bound proteins were eluted, separated by SDS-PAGE, and analyzed by Western blot using anti-FUS antibody (Proteintech, Cat No. 11570-1-AP, dilution 1:5000) to detect the enrichment of FUS protein.

Techniques: Gene Expression, Microarray, Binding Assay, RNA Binding Assay, Control

Journal: Science Progress

Article Title: Role of thrombus-derived exosomal lncRNA LOC101928697 in regulating endothelial function via FUS protein interaction in myocardial infarction

doi: 10.1177/00368504251372111

Figure Lengend Snippet: Interaction of exosomal lncRNA LOC101928697 with FUS proteins. (a and b) The western blot detection of FUS protein expression in each group of cells and the statistical graph. (c) Statistical graph of RT-qPCR to detect the expression of FUS at the mRNA level in each group of cells. (d) The fluorescence graph of fluorescence in situ hybridization (FISH) experiment. In which FUS was labeled with green fluorescence, lncRNA LOC101928697 was labeled with red fluorescence, and the nucleus was labeled with blue fluorescence (20×). (e) Western blot detection of FUS protein following RNA pull-down using sense or antisense LOC101928697 transcripts. (f) Quantification of FUS protein enrichment in sense RNA pull-down versus antisense control, based on densitometric analysis. (g-h) Western blot detection of FUS protein expression in each group of cells after knockdown or overexpression of lncRNA LOC101928697 and the statistical graphs. (i) Statistical graph of mRNA level expression of FUS in each group of cells after knockdown or overexpression of lncRNA LOC101928697 by RT-qPCR assay. a p < 0.05 compared to control group. b p < 0.05 compared to exosome group. c p < 0.05 compared to siRNA + exosome group.

Article Snippet: After extensive washing, the bound proteins were eluted, separated by SDS-PAGE, and analyzed by Western blot using anti-FUS antibody (Proteintech, Cat No. 11570-1-AP, dilution 1:5000) to detect the enrichment of FUS protein.

Techniques: Western Blot, Expressing, Quantitative RT-PCR, Fluorescence, In Situ Hybridization, Labeling, Protein Enrichment, Control, Knockdown, Over Expression

Journal: Cancer Management and Research

Article Title: Prognostic Significance of PD-L1 Expression and Its Tumor-Intrinsic Functions in Hypopharyngeal Squamous Cell Carcinoma

doi: 10.2147/CMAR.S257299

Figure Lengend Snippet: Clinicopathological Features Associated with PD-L1 Expression in 95 HSCC Patients

Article Snippet: Rabbit anti-human PD-L1 monoclonal antibody (ab205921, 1:5000, Abcam, Cambridge, MA, USA) was used for the immunohistochemistry assay.

Techniques: Expressing

Journal: Cancer Management and Research

Article Title: Prognostic Significance of PD-L1 Expression and Its Tumor-Intrinsic Functions in Hypopharyngeal Squamous Cell Carcinoma

doi: 10.2147/CMAR.S257299

Figure Lengend Snippet: PD-L1 expression in adjacent normal mucosal tissues and HSCC tissues. Representative immunohistochemical staining examples of PD-L1 protein expression are shown. In the current study there was no positive expression of PD-L1 in the mucosal tissues. Magnification, ×100 and ×200. Abbreviations: PD-L1, programmed death-ligand 1; HSCC, hypopharyngeal squamous cell carcinoma.

Article Snippet: Rabbit anti-human PD-L1 monoclonal antibody (ab205921, 1:5000, Abcam, Cambridge, MA, USA) was used for the immunohistochemistry assay.

Techniques: Expressing, Immunohistochemical staining, Staining

Journal: Cancer Management and Research

Article Title: Prognostic Significance of PD-L1 Expression and Its Tumor-Intrinsic Functions in Hypopharyngeal Squamous Cell Carcinoma

doi: 10.2147/CMAR.S257299

Figure Lengend Snippet: Kaplan-Meier analysis for overall survival of HSCC patients. Kaplan-Meier analysis of overall survival stratified by positive versus negative expression of PD-L1 in 95 HSCC patients ( P <0.001). Log rank test was used for analyzing the differences. Abbreviations: HSCC, hypopharyngeal squamous cell carcinoma; PD-L1, programmed death-ligand 1.

Article Snippet: Rabbit anti-human PD-L1 monoclonal antibody (ab205921, 1:5000, Abcam, Cambridge, MA, USA) was used for the immunohistochemistry assay.

Techniques: Expressing

Journal: Cancer Management and Research

Article Title: Prognostic Significance of PD-L1 Expression and Its Tumor-Intrinsic Functions in Hypopharyngeal Squamous Cell Carcinoma

doi: 10.2147/CMAR.S257299

Figure Lengend Snippet: Univariate and Multivariate Analyses of Clinicopathological Factors for Overall Survival in 95 Patients with HSCC

Article Snippet: Rabbit anti-human PD-L1 monoclonal antibody (ab205921, 1:5000, Abcam, Cambridge, MA, USA) was used for the immunohistochemistry assay.

Techniques: Expressing

Journal: Cancer Management and Research

Article Title: Prognostic Significance of PD-L1 Expression and Its Tumor-Intrinsic Functions in Hypopharyngeal Squamous Cell Carcinoma

doi: 10.2147/CMAR.S257299

Figure Lengend Snippet: PD-L1 promoted FaDu cells proliferation, migration and invasion in vitro. ( A and B ) Depletion of PD-L1 inhibited FaDu cells proliferation, whereas overexpression of PD-L1 stimulated FaDu cells proliferation as determined by CCK-8 assay. The data were presented as means ± standard deviation from three independent experiments. ** P <0.01. ( C and D ) The transwell migration assays showed that depletion of PD-L1 obviously inhibited the migration of FaDu cells. Conversely, overexpression of PD-L1 promoted the migration of FaDu cells. ** P <0.01. ( E and F ) The transwell invasion assays showed that depletion of PD-L1 obviously inhibited the invasion of FaDu cells. Conversely, overexpression of PD-L1 promoted the invasion of FaDu cells. The data were presented as means ± standard deviation from three independent experiments. ** P <0.01. Abbreviations: PD-L1, programmed death-ligand 1; CCK-8, Cell Counting Kit-8 assay; Cki, control siRNA; siRNA, small interfering RNA.

Article Snippet: Rabbit anti-human PD-L1 monoclonal antibody (ab205921, 1:5000, Abcam, Cambridge, MA, USA) was used for the immunohistochemistry assay.

Techniques: Migration, In Vitro, Over Expression, CCK-8 Assay, Standard Deviation, Cell Counting, Small Interfering RNA

Journal: Cancer Management and Research

Article Title: Prognostic Significance of PD-L1 Expression and Its Tumor-Intrinsic Functions in Hypopharyngeal Squamous Cell Carcinoma

doi: 10.2147/CMAR.S257299

Figure Lengend Snippet: PD-L1 promoted EMT through the Akt-mTOR signaling pathway. ( A ) FaDu cells were transfected with PD-L1 siRNA or control siRNA, expression of PD-L1, E-cadherin, N-cadherin, Vimentin, Akt, p-Akt and p-mTOR was analyzed by Western blot. β-actin was used as an inner control. ( B ) Bands of Western blot were analyzed by Image J software. Results were obtained from the ratio of target band to β-actin. The data are presented as means ± standard deviation from three independent experiments. ** P <0.01. ( C ) PD-L1-overexpressed or control FaDu cells were treated with Akt-inhibitor. The indicated protein levels were assayed by Western blot. β-actin was used as an inner control. ( D ) Bands of Western blot were analyzed by Image J software. Results were obtained from the ratio of target band to β-actin. The data are presented as means ± standard deviation from three independent experiments. * P <0.05, ** P <0.01. Abbreviations: PD-L1, programmed death-ligand 1; EMT, epithelial–mesenchymal transition; siRNA, small interfering RNA; Cki, control siRNA.

Article Snippet: Rabbit anti-human PD-L1 monoclonal antibody (ab205921, 1:5000, Abcam, Cambridge, MA, USA) was used for the immunohistochemistry assay.

Techniques: Transfection, Expressing, Western Blot, Software, Standard Deviation, Small Interfering RNA

Journal: eLife

Article Title: Nuclear m6A reader YTHDC1 promotes muscle stem cell activation/proliferation by regulating mRNA splicing and nuclear export

doi: 10.7554/elife.82703

Figure Lengend Snippet: Figure 1. m6A regulators are dynamically expressed during satellite cell (SC) lineage progression and YTHDC1 is induced upon SC activation/ proliferation. (A) Schematic illustration of SC collection from Pax7- nGFP mice. Fixed quiescent SCs (QSC), freshly isolated SCs (FISC), and cultured SCs (ASC) were subject to RNA- seq, western blotting (WB), and Immunofluorescence (IF) analyses. (B) The expression dynamics of m6A writer, reader, and eraser proteins in the above cells from analyzing the RNA- seq data. (C) Representative RNA- seq tracks showing the expression dynamics of the selected m6A regulators. (D) The expression dynamic of Ythdc1 mRNA (FPKM) from RNA- seq. (E) WB showing the induction of YTHDC1, YTHDF1, and YTHDF2 proteins upon SC activation and proliferation. * denotes the correct position of YTHDC1. Histone H3 was used as a loading control. (F) IF staining showing the induction of YTHDC1 protein upon SC activation and proliferation. Scale bar = 20 μm. (G) WB showing the predominant location of YTHDC1 in nuclear portion of C2C12 myoblasts.

Article Snippet: The following dilutions of antibodies were used for western blot staining: PAX7 (Developmental Studies Hybridoma Bank; 1:200), GAPDH (Sigma, G9545, 1:3000), YTHDC1 (Cell Signaling Technology, #77422, 1:1000), MyoD (Abclonal, 1:1000), hnRNPG (Cell Signaling Technology, #14794, 1:1000), histone H3 (Santa Cruz, sc- 517576, 1:5000), Flag- tag (Sigma, F3165, 1:3000), and THOC7 (Abclonal, A13700, 1:1000).

Techniques: Activation Assay, Isolation, Cell Culture, RNA Sequencing, Western Blot, Immunofluorescence, Expressing, Control, Staining

Journal: Cell & Bioscience

Article Title: Breaking the link between morphology and potency for mESCs

doi: 10.1186/s13578-025-01497-5

Figure Lengend Snippet: Myh9 is required for maintaining the compact morphology of mESCs. A Typical morphology of wildtype mESCs colonies. Scale bar, 50 μm. B Myh9 knockout strategy and genotype sequencing results. The mouse Myh9 gene is located on chromosome 15 and contains 41 exons. A single guide RNA (sgMyh9) was designed targeting exon 6. Following Cas9-mediated cleavage and subsequent screening, several cell clones were obtained by single-cell picking. Sanger sequencing identified two distinct mutations: one exhibited a single-base deletion near the cleavage ( Myh9 KO1), while the other exhibited a single-base insertion ( Myh9 KO2). These mutations are predicted to generate truncated mutant proteins of 217 and 229 amino acids, respectively, which correspond to only a portion of the head domain of the wildtype NMIIA. C Western blot analysis of MYH9 protein expression levels in 2 WT mESCs cell lines and 3 Myh9 knockout mESCs cell lines. GAPDH was used as loading control. D Representative morphology of Myh9 −/− mESCs. Right panel shows the enlarged images. Scale bar, 50 μm. E Representative morphology of Myh9 −/− mESCs overexpressed the control vector, full-length mouse MYH9 (mMYH9), human MYH9 (hMYH9), the N-terminal head domain of mouse MYH9 (mMYH9N) or the C-terminal tail domain of mouse MYH9 (mMYH9C). Scale bar, 250 μm.

Article Snippet: The antibodies and dilutions used were: Myosin IIA (Cell Signaling Technology, 3403, 1:1000), NANOG (Abcam, ab214549, 1:1000), OCT4 (Santa Cruz, sc-5379, 1:200), SOX2 (Proteintech, 11064-1-AP, 1:1000), KLF4 (Cell Signaling Technology, 12173, 1:1000), HRP-conjugated GAPDH (Proteintech, HRP-60004, 1:5000), HRP-labeled Goat Anti-Rabbit IgG (H + L) (Byotime, A0208, 1:2000), HRP-labeled Goat Anti-Mouse IgG (H + L) (Byotime, A0216, 1:2000).

Techniques: Knock-Out, Sequencing, Clone Assay, Mutagenesis, Western Blot, Expressing, Control, Plasmid Preparation

Journal: Cell & Bioscience

Article Title: Breaking the link between morphology and potency for mESCs

doi: 10.1186/s13578-025-01497-5

Figure Lengend Snippet: Myh9 KO mESCs have similar transcriptomic profile as WT mESCs. A Western blot analysis of MYH9, NANOG, OCT4, SOX2 and KLF4 protein expression levels in 2 WT cell lines and 2 Myh9 −/− cell lines. GAPDH was used as loading control. B Immunostaining of pluripotent marker NANOG and epithelial marker E-cadherin in WT and Myh9 −/− cell lines. Nuclear DNA was counterstained with DAPI. Scale bar, 10 μm. C Gene expression profiles of WT and Myh9 −/− mESCs as determined by RNA sequencing. Myh9 knockout has minimal impact on the expression of most naïve pluripotent markers, whereas Myh9 mRNA levels are significantly reduced

Article Snippet: The antibodies and dilutions used were: Myosin IIA (Cell Signaling Technology, 3403, 1:1000), NANOG (Abcam, ab214549, 1:1000), OCT4 (Santa Cruz, sc-5379, 1:200), SOX2 (Proteintech, 11064-1-AP, 1:1000), KLF4 (Cell Signaling Technology, 12173, 1:1000), HRP-conjugated GAPDH (Proteintech, HRP-60004, 1:5000), HRP-labeled Goat Anti-Rabbit IgG (H + L) (Byotime, A0208, 1:2000), HRP-labeled Goat Anti-Mouse IgG (H + L) (Byotime, A0216, 1:2000).

Techniques: Western Blot, Expressing, Control, Immunostaining, Marker, Gene Expression, RNA Sequencing, Knock-Out

Journal: bioRxiv

Article Title: The interferon stimulated gene-encoded protein HELZ2 inhibits human LINE-1 retrotransposition and LINE-1 RNA-mediated type I interferon induction

doi: 10.1101/2022.03.26.485892

Figure Lengend Snippet: (a) Experimental rationale for identifying host factors enriched in WT ORF1p-FLAG vs. ORF1p-FLAG (M8 [RBM]) immunoprecipitation reactions. Hypothetical diagrams of the proteins associating with WT and M8 (RBM) mutant RNP particles. Green circles, ORF1p-FLAG. Blue Oval, ORF2p. Red circle, purple squared oval, and green rectangle, host factors that might associate with ORF1p-FLAG and/or L1 RNPs. (b) The ORF1p (M8 [RBM]) mutant does not efficiently interact with Poly(A) Binding Protein Cytoplasmic 1 (PABPC1) . HeLa-JVM cells were transfected with either pJM101/L1.3 (no FLAG), pJM101/L1.3-FLAG (WT ORF1p-FLAG), or pALAF008 (ORF1p-FLAG [M8 [RBM]] mutant). An anti-FLAG antibody was used to immunoprecipitate ORF1p-FLAG. Western blots detected ORF1p (anti-FLAG), PABPC1 (anti-PABC1), and GAPDH (anti-GAPDH) in the input and IP fractions. GAPDH served as a loading control for the input fractions and a negative control in the IP experiments. (c) Separation of proteins associated with the WT and mutant ORF1p-FLAG proteins. The WT and M8 (RBM) mutant ORF1p-FLAG IP complexes were separated by SDS-PAGE using a 4-15% gradient gel and silver staining visualized the proteins. Protein size standards (kDa) are shown at the left of the gel. Black arrowhead, the expected molecular weight of ORF1p-FLAG. (d) Gene Ontology (GO) analysis identifies cellular proteins enriched in IP WT ORF1p-FLAG vs. the mutant ORF1p-FLAG complex. Cellular proteins present in the WT ORF1p and (M8 [RBM])-FLAG mutant IP complexes were identified using LC-MS/MS. Proteins having at least five peptide matches to the UniProt database ( https://www.uniprot.org/ ) were subjected to PANTHER statistical enrichment analysis. The top 10 GO terms with the lowest false discovery rates (FDRs) are sorted in descending values. X-axis, −log10 FDR. Y-axis, GO term. Red lettering, viral related GO terms. (e) Leading Edge Analysis identifies interferon-related gene sets enriched upon WT ORF1p-FLAG immunoprecipitation. Gene Set Enrichment Analysis (GSEA) of peptides immunoprecipitated in WT ORF1p-FLAG vs. (M8 [RBM])-FLAG IP complexes was performed using hallmark gene sets in the Molecular Signatures Database ( MSigDB: www.gsea-msigdb.org/gsea/msigdb/ ), followed by Leading Edge Analysis to determine gene set enrichment scores. The top five hallmark gene sets with the highest normalized enrichment score (NES) are sorted in descending values. X-axis, NES. Y-axis, hallmark gene sets. (f) The expression of engineered L1s modestly up-regulates IFN-α expression. HEK293T were transfected with either pCEP4 (an empty vector control), pJM101/L1.3FLAG (WT), pJM105/L1.3 (RT-), or pALAF008 (M8 [RBM]). RT-qPCR was used to quantify IFN-α (primer set: IFN-α) and L1 expression (primer set: mneoI [Alu or L1]) ~96 hours post-transfection. IFN-α and L1 expression levels were normalized using β-actin ( ACTB ) as a control (primer set: Beta-actin). X-axis, name of constructs. Control, pCEP4. Y-axis, relative RNA expression levels normalized to the pCEP4 empty vector control. Red bars, normalized IFN-α expression levels. Gray bars, normalized L1 expression levels. Values from three independent biological replicates ± SEM are depicted in the graph. The p -values were calculated using a one-way ANOVA followed by Bonferroni-Holm post-hoc tests: pairwise comparisons of IFN-α relative to the pCEP4 control, p = 0.00028*** (WT); 0.00011*** (RT-); 3.14 × 10 −6 *** (M8 [RBM]). Pairwise comparisons of IFN-α: WT vs. RT-, p = 0.21 ns ; WT vs. M8 (RBM), p = 0.00036***. Pairwise comparisons of L1 relative to WT, p = 0.87 ns (RT-), p = 0.10 ns (M8 [RBM]); ns: not significant; *** p <0.001.

Article Snippet: Mouse monoclonal anti-FLAG M2 antibody (1/5000), (Sigma-Aldrich, F1804, RRID: AB_262044) Mouse monoclonal anti-MYC antibody (1/5000), (Cell Signaling Technology, 9B11, RRID: AB_331783) Rabbit polyclonal anti-PABPC1 antibody (1/5000), (Abcam, ab21060, RRID: AB_777008) Mouse monoclonal anti-GAPDH antibody (1/5000), (Millipore, MAB374, RRID: AB_2107445) Mouse monoclonal anti-Actin antibody (1/5000), (Millipore, MAB1501, RRID: AB_2223041) Rabbit polyclonal anti-T7-tag antibody (1/5000), (Cell Signaling Technology, D9E1X, RRID: AB_2798161) Goat polyclonal anti-Luciferase antibody (1/2000), (Promega, G7451, RRID: AB_430862) Mouse monoclonal anti-ORF1p (4H1) antibody (1/2000), (Millipore, MABC1152) Mouse monoclonal anti-eIF3 p110 (B-6) antibody (1/5000), (Santa Cruz Biotechnology, sc-74507, RRID: AB_1122487)

Techniques: Immunoprecipitation, Mutagenesis, Binding Assay, Transfection, Western Blot, Negative Control, SDS Page, Silver Staining, Molecular Weight, Liquid Chromatography with Mass Spectroscopy, Expressing, Plasmid Preparation, Quantitative RT-PCR, Construct, RNA Expression